Method of loading a crystallization device

Abstract

The present invention pertains to a method for loading a crystallization device and for manufacturing a crystallization device comprising multiple receptacles with a pre-defined amount of at least one matrix-forming compound capable of forming a crystallization matrix for a membrane protein, said method comprising the following steps: a) Modifying the state of aggregation of said at least one matrix-forming compound to a fluidic state which allows dispensing said at least one matrix-forming compound, and b) dispensing a defined amount of said at least one matrix-forming compound into at least one receptacle of the crystallization device, wherein said dispensed matrix-forming compound solidifies within said receptacle. Thereby, pre-filled crystallization devices are obtained which can be used as consumables in particular in automated crystallization processes. Also provided are protein crystallization methods using respectively prepared crystallization devices.

The invention claimed is: 
     
       1. A method for loading a crystallization device comprising multiple receptacles with a pre-defined amount of at least one matrix-forming compound capable of forming a crystallization matrix for a membrane protein, comprising:
 a) modifying the state of aggregation of said at least one matrix-forming compound from a solid to a fluidic state which allows dispensing said at least one matrix-forming compound; 
 b) dispensing a defined amount of said at least one matrix-forming compound into at least one receptacle of the crystallization device, wherein the at least one matrix-forming compound does not comprise the membrane protein; and 
 c) solidifying said at least one matrix-forming compound in said receptacle to form a solidified matrix-forming compound, wherein the solidified matrix-forming compound does not comprise the membrane protein and has not formed a crystallization matrix. 
 
     
     
       2. The method according to  claim 1 , wherein the state of aggregation of said at least one matrix-forming compound is modified by modifying the temperature. 
     
     
       3. The method according to  claim 1 , wherein dispensing said at least one matrix-forming compound into at least one receptacle is automated by using a robotic system comprising at least one dispensing unit. 
     
     
       4. The method according to  claim 1 , wherein said at least one matrix-forming compound:
 a) is capable of forming a meso phase; and/or 
 b) is capable of forming a cubic phase; and/or 
 c) is capable of forming a sponge phase; and/or 
 d) is a lipidic compound; and/or 
 e) is amphiphilic; and/or 
 f) comprises a saturated or unsaturated fatty-acid chain; and/or 
 g) is an alcohol derivative from a fatty acid. 
 
     
     
       5. The method according to  claim 1 , wherein:
 a) said at least one matrix-forming compo is mixed with an additive thereby forming an additive composition; and/or 
 b) said at least one matrix-forming compound or said additive composition comprises:
 i) at least one compound selected from the group consisting of fatty acids, alcohol derivatives from fatty acids, monoglycerides, diglycerides, lipids and their derivatives, the corresponding compounds that have their acid group(s) replaced by a hydroxyl or thiol or ether or thioether group or ω-hydroxyalkenes or their ethers or homologous thiols or thioethers; monoacylglycerols, cis monounsaturated monoacylglycerols, monoolein (C18: c9), monopalmitolein (C16: c9) and monovacennin (C18: c7); medium-chain length alkyl glycosides; polyalkylenglycols, polyethylenglycols, diacylglycerophospholipids, monoacylglycerophospholipids and derivatives thereof capable of forming a crystallization matrix for the membrane protein; and/or 
 ii)at least one compound selected from the group consisting of polyketides, saccharelipids, prenol lipids, sterol lipids, sphingolipids, glycerophospholipids and glycerolipids and/or derivates of lipids, phosphatidylcholine (PC), DOPC, phosphatidylethanolamine, DOPE, phosphatidylserine, DOPS, cardiolipin, lyso-phosphatidylcholine, 2-monoolein, oleamide, cholesterol, cell membrane components, and natural or synthetic compounds stabilizing the membrane protein in the crystallization matrix. 
 
 
     
     
       6. A method for manufacturing a crystallization device comprising multiple receptacles with a pre-defined amount of at least one matrix-forming compound capable of forming a crystallization matrix for a membrane protein, wherein the method for manufacturing a clystallization device comprises the method for loading a crystallization device according to  claim 1 . 
     
     
       7. The method according to  claim 1 , wherein modifying the state of aggregation of said at least one matrix-forming compound comprises heating said at least one matrix-forming compound. 
     
     
       8. The method of  claim 3 , wherein the method is automated.